Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Jan;17(1):219–229. doi: 10.1128/mcb.17.1.219

Lack of gene- and strand-specific DNA repair in RNA polymerase III-transcribed human tRNA genes.

R Dammann 1, G P Pfeifer 1
PMCID: PMC231746  PMID: 8972202

Abstract

UV light induces DNA lesions which are removed by nucleotide excision repair. Genes transcribed by RNA polymerase II are repaired faster than the flanking chromatin, and the transcribed strand is repaired faster than the coding strand. Transcription-coupled repair is not seen in RNA polymerase I-transcribed human rRNA genes. Since repair of genes transcribed by RNA polymerase III has not been analyzed before, we investigated DNA repair of tRNA genes after irradiation of human fibroblasts with UVC. We studied the repair of UV-induced cyclobutane pyrimidine dimers at nucleotide resolution by ligation-mediated PCR. A single-copy gene encoding selenocysteine tRNA, a tRNA valine gene, and their flanking sequences were analyzed. Protein-DNA footprinting showed that both genes were occupied by regulatory factors in vivo, and Northern blotting and nuclear run-on analysis of the tRNA indicated that these genes were actively transcribed. We found that both genes were repaired slower than RNA polymerase II-transcribed genes. No major difference between repair of the transcribed and the coding DNA strands was detected. Transcribed sequences of the tRNA genes were not repaired faster than flanking sequences. Indeed, several sequence positions in the 5' flanking region of the tRNA(Val) gene were repaired more efficiently than the gene itself. These results indicate that unlike RNA polymerase II, RNA polymerase III has no stimulatory effect on DNA repair. Since tRNA genes are covered by the regulatory factor TFIIIC and RNA polymerase III, these proteins may actually inhibit the DNA's accessibility to repair enzymes.

Full Text

The Full Text of this article is available as a PDF (636.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Armstrong J. D., Kunz B. A. Excision repair influences the site and strand specificity of sunlight mutagenesis in yeast. Mutat Res. 1992 Aug;274(2):123–133. doi: 10.1016/0921-8777(92)90059-c. [DOI] [PubMed] [Google Scholar]
  2. Armstrong J. D., Kunz B. A. Site and strand specificity of UVB mutagenesis in the SUP4-o gene of yeast. Proc Natl Acad Sci U S A. 1990 Nov;87(22):9005–9009. doi: 10.1073/pnas.87.22.9005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bohr V. A. DNA repair and transcriptional activity in genes. J Cell Sci. 1988 Oct;91(Pt 2):175–178. doi: 10.1242/jcs.91.2.175. [DOI] [PubMed] [Google Scholar]
  4. Bohr V. A., Okumoto D. S., Hanawalt P. C. Survival of UV-irradiated mammalian cells correlates with efficient DNA repair in an essential gene. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3830–3833. doi: 10.1073/pnas.83.11.3830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bohr V. A., Smith C. A., Okumoto D. S., Hanawalt P. C. DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell. 1985 Feb;40(2):359–369. doi: 10.1016/0092-8674(85)90150-3. [DOI] [PubMed] [Google Scholar]
  6. Brash D. E., Seetharam S., Kraemer K. H., Seidman M. M., Bredberg A. Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3782–3786. doi: 10.1073/pnas.84.11.3782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brash D. E. UV mutagenic photoproducts in Escherichia coli and human cells: a molecular genetics perspective on human skin cancer. Photochem Photobiol. 1988 Jul;48(1):59–66. doi: 10.1111/j.1751-1097.1988.tb02786.x. [DOI] [PubMed] [Google Scholar]
  8. Carbon P., Krol A. Transcription of the Xenopus laevis selenocysteine tRNA(Ser)Sec gene: a system that combines an internal B box and upstream elements also found in U6 snRNA genes. EMBO J. 1991 Mar;10(3):599–606. doi: 10.1002/j.1460-2075.1991.tb07987.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Christians F. C., Hanawalt P. C. Lack of transcription-coupled repair in mammalian ribosomal RNA genes. Biochemistry. 1993 Oct 5;32(39):10512–10518. doi: 10.1021/bi00090a030. [DOI] [PubMed] [Google Scholar]
  10. Clarke E. M., Peterson C. L., Brainard A. V., Riggs D. L. Regulation of the RNA polymerase I and III transcription systems in response to growth conditions. J Biol Chem. 1996 Sep 6;271(36):22189–22195. doi: 10.1074/jbc.271.36.22189. [DOI] [PubMed] [Google Scholar]
  11. Craig L. C., Wang L. P., Lee M. M., Pirtle I. L., Pirtle R. M. A human tRNA gene cluster encoding the major and minor valine tRNAs and a lysine tRNA. DNA. 1989 Sep;8(7):457–471. doi: 10.1089/dna.1.1989.8.457. [DOI] [PubMed] [Google Scholar]
  12. Dieci G., Sentenac A. Facilitated recycling pathway for RNA polymerase III. Cell. 1996 Jan 26;84(2):245–252. doi: 10.1016/s0092-8674(00)80979-4. [DOI] [PubMed] [Google Scholar]
  13. Drapkin R., Reardon J. T., Ansari A., Huang J. C., Zawel L., Ahn K., Sancar A., Reinberg D. Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II. Nature. 1994 Apr 21;368(6473):769–772. doi: 10.1038/368769a0. [DOI] [PubMed] [Google Scholar]
  14. Fritz L. K., Smerdon M. J. Repair of UV damage in actively transcribed ribosomal genes. Biochemistry. 1995 Oct 10;34(40):13117–13124. doi: 10.1021/bi00040a024. [DOI] [PubMed] [Google Scholar]
  15. Gao S., Drouin R., Holmquist G. P. DNA repair rates mapped along the human PGK1 gene at nucleotide resolution. Science. 1994 Mar 11;263(5152):1438–1440. doi: 10.1126/science.8128226. [DOI] [PubMed] [Google Scholar]
  16. Geiduschek E. P., Kassavetis G. A. Comparing transcriptional initiation by RNA polymerases I and III. Curr Opin Cell Biol. 1995 Jun;7(3):344–351. doi: 10.1016/0955-0674(95)80089-1. [DOI] [PubMed] [Google Scholar]
  17. Geiduschek E. P., Tocchini-Valentini G. P. Transcription by RNA polymerase III. Annu Rev Biochem. 1988;57:873–914. doi: 10.1146/annurev.bi.57.070188.004301. [DOI] [PubMed] [Google Scholar]
  18. Hanawalt P. C., Donahue B. A., Sweder K. S. Repair and transcription. Collision or collusion? Curr Biol. 1994 Jun 1;4(6):518–521. doi: 10.1016/s0960-9822(00)00112-3. [DOI] [PubMed] [Google Scholar]
  19. Heider J., Leinfelder W., Böck A. Occurrence and functional compatibility within Enterobacteriaceae of a tRNA species which inserts selenocysteine into protein. Nucleic Acids Res. 1989 Apr 11;17(7):2529–2540. doi: 10.1093/nar/17.7.2529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Huet J., Sentenac A. The TATA-binding protein participates in TFIIIB assembly on tRNA genes. Nucleic Acids Res. 1992 Dec 25;20(24):6451–6454. doi: 10.1093/nar/20.24.6451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Joazeiro C. A., Kassavetis G. A., Geiduschek E. P. Alternative outcomes in assembly of promoter complexes: the roles of TBP and a flexible linker in placing TFIIIB on tRNA genes. Genes Dev. 1996 Mar 15;10(6):725–739. doi: 10.1101/gad.10.6.725. [DOI] [PubMed] [Google Scholar]
  22. Kantor G. J., Setlow R. B. Rate and extent of DNA repair in nondividing human diploid fibroblasts. Cancer Res. 1981 Mar;41(3):819–825. [PubMed] [Google Scholar]
  23. Lee B. J., Rajagopalan M., Kim Y. S., You K. H., Jacobson K. B., Hatfield D. Selenocysteine tRNA[Ser]Sec gene is ubiquitous within the animal kingdom. Mol Cell Biol. 1990 May;10(5):1940–1949. doi: 10.1128/mcb.10.5.1940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee B. J., Worland P. J., Davis J. N., Stadtman T. C., Hatfield D. L. Identification of a selenocysteyl-tRNA(Ser) in mammalian cells that recognizes the nonsense codon, UGA. J Biol Chem. 1989 Jun 15;264(17):9724–9727. [PubMed] [Google Scholar]
  25. Leinfelder W., Zehelein E., Mandrand-Berthelot M. A., Böck A. Gene for a novel tRNA species that accepts L-serine and cotranslationally inserts selenocysteine. Nature. 1988 Feb 25;331(6158):723–725. doi: 10.1038/331723a0. [DOI] [PubMed] [Google Scholar]
  26. Low S. C., Berry M. J. Knowing when not to stop: selenocysteine incorporation in eukaryotes. Trends Biochem Sci. 1996 Jun;21(6):203–208. [PubMed] [Google Scholar]
  27. Mauck J. C., Green H. Regulation of pre-transfer RNA synthesis during transition from resting to growing state. Cell. 1974 Oct;3(2):171–177. doi: 10.1016/0092-8674(74)90122-6. [DOI] [PubMed] [Google Scholar]
  28. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  29. McBride O. W., Rajagopalan M., Hatfield D. Opal suppressor phosphoserine tRNA gene and pseudogene are located on human chromosomes 19 and 22, respectively. J Biol Chem. 1987 Aug 15;262(23):11163–11166. [PubMed] [Google Scholar]
  30. Mellon I., Spivak G., Hanawalt P. C. Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell. 1987 Oct 23;51(2):241–249. doi: 10.1016/0092-8674(87)90151-6. [DOI] [PubMed] [Google Scholar]
  31. Mitchell D. L., Nairn R. S. The biology of the (6-4) photoproduct. Photochem Photobiol. 1989 Jun;49(6):805–819. doi: 10.1111/j.1751-1097.1989.tb05578.x. [DOI] [PubMed] [Google Scholar]
  32. Myslinski E., Schuster C., Huet J., Sentenac A., Krol A., Carbon P. Point mutations 5' to the tRNA selenocysteine TATA box alter RNA polymerase III transcription by affecting the binding of TBP. Nucleic Acids Res. 1993 Dec 25;21(25):5852–5858. doi: 10.1093/nar/21.25.5852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. O'Neill G. P., Söll D. Expression of the Synechocystis sp. strain PCC 6803 tRNA(Glu) gene provides tRNA for protein and chlorophyll biosynthesis. J Bacteriol. 1990 Nov;172(11):6363–6371. doi: 10.1128/jb.172.11.6363-6371.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. O'Neill V. A., Eden F. C., Pratt K., Hatfield D. L. A human opal suppressor tRNA gene and pseudogene. J Biol Chem. 1985 Feb 25;260(4):2501–2508. [PubMed] [Google Scholar]
  35. Pfeifer G. P., Drouin R., Holmquist G. P. Detection of DNA adducts at the DNA sequence level by ligation-mediated PCR. Mutat Res. 1993 Jul;288(1):39–46. doi: 10.1016/0027-5107(93)90206-u. [DOI] [PubMed] [Google Scholar]
  36. Pfeifer G. P., Drouin R., Riggs A. D., Holmquist G. P. Binding of transcription factors creates hot spots for UV photoproducts in vivo. Mol Cell Biol. 1992 Apr;12(4):1798–1804. doi: 10.1128/mcb.12.4.1798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pfeifer G. P., Drouin R., Riggs A. D., Holmquist G. P. In vivo mapping of a DNA adduct at nucleotide resolution: detection of pyrimidine (6-4) pyrimidone photoproducts by ligation-mediated polymerase chain reaction. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1374–1378. doi: 10.1073/pnas.88.4.1374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pfeifer G. P., Singer-Sam J., Riggs A. D. Analysis of methylation and chromatin structure. Methods Enzymol. 1993;225:567–583. doi: 10.1016/0076-6879(93)25037-3. [DOI] [PubMed] [Google Scholar]
  39. Rychlik W., Rhoads R. E. A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Res. 1989 Nov 11;17(21):8543–8551. doi: 10.1093/nar/17.21.8543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sage E. Distribution and repair of photolesions in DNA: genetic consequences and the role of sequence context. Photochem Photobiol. 1993 Jan;57(1):163–174. doi: 10.1111/j.1751-1097.1993.tb02273.x. [DOI] [PubMed] [Google Scholar]
  41. Saluz H. P., Jost J. P. Approaches to characterize protein-DNA interactions in vivo. Crit Rev Eukaryot Gene Expr. 1993;3(1):1–29. [PubMed] [Google Scholar]
  42. Schaeffer L., Roy R., Humbert S., Moncollin V., Vermeulen W., Hoeijmakers J. H., Chambon P., Egly J. M. DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor. Science. 1993 Apr 2;260(5104):58–63. doi: 10.1126/science.8465201. [DOI] [PubMed] [Google Scholar]
  43. Schmutzler C., Gross H. J. Genes, variant genes, and pseudogenes of the human tRNA(Val) gene family are differentially expressed in HeLa cells and in human placenta. Nucleic Acids Res. 1990 Sep 11;18(17):5001–5008. doi: 10.1093/nar/18.17.5001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Seetharam S., Kraemer K. H., Waters H. L., Seidman M. M. Ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum lymphoblastoid cells and fibroblasts. Mutat Res. 1991 Jan;254(1):97–105. doi: 10.1016/0921-8777(91)90045-q. [DOI] [PubMed] [Google Scholar]
  45. Selby C. P., Sancar A. Mechanisms of transcription-repair coupling and mutation frequency decline. Microbiol Rev. 1994 Sep;58(3):317–329. doi: 10.1128/mr.58.3.317-329.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Selby C. P., Sancar A. Molecular mechanism of transcription-repair coupling. Science. 1993 Apr 2;260(5104):53–58. doi: 10.1126/science.8465200. [DOI] [PubMed] [Google Scholar]
  47. Shortridge R. D., Johnson G. D., Craig L. C., Pirtle I. L., Pirtle R. M. A human tRNA gene heterocluster encoding threonine, proline and valine tRNAs. Gene. 1989 Jul 15;79(2):309–324. doi: 10.1016/0378-1119(89)90213-8. [DOI] [PubMed] [Google Scholar]
  48. Smerdon M. J. DNA repair and the role of chromatin structure. Curr Opin Cell Biol. 1991 Jun;3(3):422–428. doi: 10.1016/0955-0674(91)90069-b. [DOI] [PubMed] [Google Scholar]
  49. Sprague K. U. New twists in class III transcription. Curr Opin Cell Biol. 1992 Jun;4(3):475–479. doi: 10.1016/0955-0674(92)90014-4. [DOI] [PubMed] [Google Scholar]
  50. Stevnsner T., May A., Petersen L. N., Larminat F., Pirsel M., Bohr V. A. Repair of ribosomal RNA genes in hamster cells after UV irradiation, or treatment with cisplatin or alkylating agents. Carcinogenesis. 1993 Aug;14(8):1591–1596. doi: 10.1093/carcin/14.8.1591. [DOI] [PubMed] [Google Scholar]
  51. Thomann H. U., Schmutzler C., Hüdepohl U., Blow M., Gross H. J. Genes, variant genes and pseudogenes of the human tRNA(Val) gene family. Expression and pre-tRNA maturation in vitro. J Mol Biol. 1989 Oct 20;209(4):505–523. doi: 10.1016/0022-2836(89)90590-1. [DOI] [PubMed] [Google Scholar]
  52. Tommasi S., Pfeifer G. P. In vivo structure of the human cdc2 promoter: release of a p130-E2F-4 complex from sequences immediately upstream of the transcription initiation site coincides with induction of cdc2 expression. Mol Cell Biol. 1995 Dec;15(12):6901–6913. doi: 10.1128/mcb.15.12.6901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tornaletti S., Pfeifer G. P. Slow repair of pyrimidine dimers at p53 mutation hotspots in skin cancer. Science. 1994 Mar 11;263(5152):1436–1438. doi: 10.1126/science.8128225. [DOI] [PubMed] [Google Scholar]
  54. Tornaletti S., Pfeifer G. P. UV damage and repair mechanisms in mammalian cells. Bioessays. 1996 Mar;18(3):221–228. doi: 10.1002/bies.950180309. [DOI] [PubMed] [Google Scholar]
  55. Tornaletti S., Pfeifer G. P. UV light as a footprinting agent: modulation of UV-induced DNA damage by transcription factors bound at the promoters of three human genes. J Mol Biol. 1995 Jun 16;249(4):714–728. doi: 10.1006/jmbi.1995.0331. [DOI] [PubMed] [Google Scholar]
  56. Troelstra C., van Gool A., de Wit J., Vermeulen W., Bootsma D., Hoeijmakers J. H. ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne's syndrome and preferential repair of active genes. Cell. 1992 Dec 11;71(6):939–953. doi: 10.1016/0092-8674(92)90390-x. [DOI] [PubMed] [Google Scholar]
  57. Tu Y., Tornaletti S., Pfeifer G. P. DNA repair domains within a human gene: selective repair of sequences near the transcription initiation site. EMBO J. 1996 Feb 1;15(3):675–683. [PMC free article] [PubMed] [Google Scholar]
  58. Törmänen V. T., Pfeifer G. P. Mapping of UV photoproducts within ras proto-oncogenes in UV-irradiated cells: correlation with mutations in human skin cancer. Oncogene. 1992 Sep;7(9):1729–1736. [PubMed] [Google Scholar]
  59. Verhage R. A., Van de Putte P., Brouwer J. Repair of rDNA in Saccharomyces cerevisiae: RAD4-independent strand-specific nucleotide excision repair of RNA polymerase I transcribed genes. Nucleic Acids Res. 1996 Mar 15;24(6):1020–1025. doi: 10.1093/nar/24.6.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Vos J. M., Wauthier E. L. Differential introduction of DNA damage and repair in mammalian genes transcribed by RNA polymerases I and II. Mol Cell Biol. 1991 Apr;11(4):2245–2252. doi: 10.1128/mcb.11.4.2245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Vrieling H., Van Rooijen M. L., Groen N. A., Zdzienicka M. Z., Simons J. W., Lohman P. H., van Zeeland A. A. DNA strand specificity for UV-induced mutations in mammalian cells. Mol Cell Biol. 1989 Mar;9(3):1277–1283. doi: 10.1128/mcb.9.3.1277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. White R. J., Gottlieb T. M., Downes C. S., Jackson S. P. Cell cycle regulation of RNA polymerase III transcription. Mol Cell Biol. 1995 Dec;15(12):6653–6662. doi: 10.1128/mcb.15.12.6653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Willis I. M. RNA polymerase III. Genes, factors and transcriptional specificity. Eur J Biochem. 1993 Feb 15;212(1):1–11. doi: 10.1111/j.1432-1033.1993.tb17626.x. [DOI] [PubMed] [Google Scholar]
  64. Wright S., Bishop J. M. DNA sequences that mediate attenuation of transcription from the mouse protooncogene myc. Proc Natl Acad Sci U S A. 1989 Jan;86(2):505–509. doi: 10.1073/pnas.86.2.505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. van Hoffen A., Venema J., Meschini R., van Zeeland A. A., Mullenders L. H. Transcription-coupled repair removes both cyclobutane pyrimidine dimers and 6-4 photoproducts with equal efficiency and in a sequential way from transcribed DNA in xeroderma pigmentosum group C fibroblasts. EMBO J. 1995 Jan 16;14(2):360–367. doi: 10.1002/j.1460-2075.1995.tb07010.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES